Although subtypes of pancreatic ductal adenocarcinoma (PDAC) were described, this malignancy is clinically still treated as a single disease. Here, we present patient-derived models representing the full spectrum of previously identified quasi-mesenchymal (QM-PDA), classical and exocrine-like PDAC subtypes, and identify two markers—HNF1A and KRT81—that enable stratification of tumors into different subtypes by immunohistochemistry. Individuals bearing tumors of these subtypes show significant differences in overall survival and their tumors differ in drug sensitivity, with the exocrine-like subtype being resistant to tyrosine kinase inhibitors and paclitaxel. Cytochrome P450 3A5 (CYP3A5) metabolizes these compounds in tumors of the exocrine-like subtype, and pharmacological or shRNA-mediated CYP3A5 inhibition sensitizes tumor cells to these drugs. Whereas hepatocyte nuclear factor 4 alpha (HNF4A) controls basal expression of CYP3A5, drug-induced CYP3A5 upregulation is mediated by the nuclear receptor NR1I2. CYP3A5 also contributes to acquired drug resistance in QM-PDA and classical PDAC, and is highly expressed in several additional malignancies. These findings designate CYP3A5 as predictor of therapy response and as a tumor cell-autonomous detoxification mechanism that must be overcome to prevent drug resistance.

Activating mutations in the proto-oncogene KRAS are a hallmark of pancreatic ductal adenocarcinoma (PDAC), an aggressive malignancy with few effective therapeutic options. Despite efforts to develop KRAS-targeted drugs, the absolute dependence of PDAC cells on KRAS remains incompletely understood. Here we model complete KRAS inhibition using CRISPR/Cas-mediated genome editing and demonstrate that KRAS is dispensable in a subset of human and mouse PDAC cells. Remarkably, nearly all KRAS deficient cells exhibit phosphoinositide 3-kinase (PI3K)-dependent mitogen-activated protein kinase (MAPK) signaling and induced sensitivity to PI3K inhibitors. Furthermore, comparison of gene expression profiles of PDAC cells retaining or lacking KRAS reveal a role of KRAS in the suppression of metastasis-related genes. Collectively, these data underscore the potential for PDAC resistance to even the very best KRAS inhibitors and provide insights into mechanisms of response and resistance to KRAS inhibition.

SummaryThe influence of the gut microbiome on metabolic and behavioral traits is widely accepted, though the microbiome-derived metabolites involved remain unclear. We carried out untargeted urine 1H-NMR spectroscopy-based metabolic phenotyping in an isogenic C57BL/6J mouse population (n = 50) and show that microbial-host co-metabolites are prodromal (i.e., early) markers predicting future divergence in metabolic (obesity and glucose homeostasis) and behavioral (anxiety and activity) outcomes with 94%–100% accuracy. Some of these metabolites also modulate disease phenotypes, best illustrated by trimethylamine-N-oxide (TMAO), a product of microbial-host co-metabolism predicting future obesity, impaired glucose tolerance (IGT), and behavior while reducing endoplasmic reticulum stress and lipogenesis in 3T3-L1 adipocytes. Chronic in vivo TMAO treatment limits IGT in HFD-fed mice and isolated pancreatic islets by increasing insulin secretion. We highlight the prodromal potential of microbial metabolites to predict disease outcomes and their potential in shaping mammalian phenotypic heterogeneity.

KRAS is one of the most frequently mutated oncogenes in human cancer. Despite substantial efforts, no clinically applicable strategy has yet been developed to effectively treat KRAS-mutant tumors. Here, we perform a cell-line-based screen and identify strong synergistic interactions between cell-cycle checkpoint-abrogating Chk1- and MK2 inhibitors, specifically in KRAS- and BRAF-driven cells. Mechanistically, we show that KRAS-mutant cancer displays intrinsic genotoxic stress, leading to tonic Chk1- and MK2 activity. We demonstrate that simultaneous Chk1- and MK2 inhibition leads to mitotic catastrophe in KRAS-mutant cells. This actionable synergistic interaction is validated using xenograft models, as well as distinct Kras- or Braf-driven autochthonous murine cancer models. Lastly, we show that combined checkpoint inhibition induces apoptotic cell death in KRAS- or BRAF-mutant tumor cells directly isolated from patients. These results strongly recommend simultaneous Chk1- and MK2 inhibition as a therapeutic strategy for the treatment of KRAS- or BRAF-driven cancers.

SummaryMethods to isolate and culture primary prostate epithelial stem/progenitor cells (PESCs) have proven difficult and ineffective. Here, we present a method to grow and expand both murine and human basal PESCs long term in serum- and feeder-free conditions. The method enriches for adherent mouse basal PESCs with a Lin−SCA-1+CD49f+TROP2high phenotype. Progesterone and sodium selenite are additionally required for the growth of human Lin−CD49f+TROP2high PESCs. The gene-expression profiles of expanded basal PESCs show similarities to ESCs, and NF-kB function is critical for epithelial differentiation of sphere-cultured PESCs. When transplanted in combination with urogenital sinus mesenchyme, expanded mouse and human PESCs generate ectopic prostatic tubules, demonstrating their stem cell activity in vivo. This novel method will facilitate the molecular, genomic, and functional characterization of normal and pathologic prostate glands of mouse and human origin.

Cell 162, 146-159; July 2, 2015) Our paper presented a new algorithm, named PreCISE, designed to identify synergistic drug interactions that are effective at killing cancer cells harboring specific driver mutations. Using this platform and a cell-line-based screen, we identified a synergistic drug interaction between Chk1-and MK2 inhibitors in KRASor BRAF-driven cells, and that combination of therapy focused on these two kinase inhibitors is effective at inducing cell death of KRASand BRAF mutant tumors in vivo.While reviewing the paper after publication, we noticed that we had included two erroneous duplications of western blot loading control bands in the final version of Figure 2E. This figure shows that simultaneous inhibition of Chk1 and MK2 induces genotoxic stress and apoptosis in several KRAS-driven cancer cell lines and respective controls. The incorrect loading controls are presented for the HSP27 blot for the H1703 cell line, as well as for the CDC25B blot for the H1437 cell line. The errors occurred when we were copying each blot to construct the final figure. We recovered the original autoradiographs of these experiments and now provide a new version of the figure containing the correct loading controls. The correct data supports the original interpretation of the experiment, and the conclusions of the paper remain unchanged. In addition, we observed a typo in Figure S3A, showing the distribution of all cell lines used in the initial screen of our paper. In the pie chart, the slice of the pie in purple representing ''lung squamous'' cell lines was incorrectly labeled with n = 18. The correct value is n = 3, as depicted in the figure legend and in the main text. Both figures are now corrected online. We regret not being able to identify these errors before and sincerely apologize for any inconvenience they may have caused.

The study reports the identification and validation of novel PDAC biomarkers relevant for the development of patient stratification tools. In addition, cadherin-17 and galectin-4 may serve as targets for bispecific antibodies as novel therapeutics in PDAC.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with poor prognosis. Treatment with gemcitabine, the FOLFIRINOX scheme or nab-paclitaxel offer only a modest increase in overall survival. For a number of other carcinomas, tumor subtypes have been uncovered that allow the use of targeted therapies. Although subtypes of PDAC were described, this malignancy is clinically still treated as a single disease. We established patient-derived models representing the full spectrum of previously identified quasi-mesenchymal (QM-PDA), classical and exocrine-like PDAC subtypes, and identified two markers—HNF1A and KRT81—that enable stratification of tumors into different subtypes by immunohistochemistry. Patients bearing tumors of these subtypes show significant differences in overall survival and their tumors differ in drug sensitivity, with the exocrine-like subtype being resistant to tyrosine kinase inhibitors and paclitaxel. The xenobiotic enzyme, cytochrome P450 3A5 (CYP3A5), metabolizes these compounds in tumor cells of the exocrine-like subtype, and pharmacological or short hairpin RNA (shRNA)-mediated CYP3A5 inhibition sensitizes tumor cells to these drugs. Additionally, retrospective analysis of a large patient cohort confirmed that CYP3A5 is predominantly found in those patient tumors classified as exocrine-like (Noll, Eisen et al., Nature Medicine (2016) accepted). Whereas the hepatocyte nuclear factor 4, alpha (HNF4A) controls basal expression of CYP3A5, drug-induced CYP3A5 upregulation is mediated by the nuclear receptor NR1I2. Interfering with these regulatory mechanisms may provide an alternative approach to suppress the CYP3A5 mediated resistance pathway. CYP3A5 also contributes to acquired drug resistance in QM-PDA and classical PDAC in vitro and in vivo. Finally, CYP3A5 is highly expressed in several additional malignancies including hepatocellular and cervical carcinomas raising the possibility that the CYP3A5 resistance mechanism is operational in a variety of human cancers. These findings designate CYP3A5 as predictor of therapy response and as a tumor cell-autonomous detoxification mechanism that must be overcome to prevent drug resistance. Citation Format: Elisa M. Noll, Christian Eisen, Albrecht Stenzinger, Elisa Espinet, Alexander Muckenhuber, Corinna Klein, Vanessa Vogel, Bernd Klaus, Wiebke Nadler, Christoph Rösli, Christian Lutz, Michael Kulke, Jan Engelhardt, Franziska Zickgraf, Octavio Espinosa, Matthias Schlesner, Xiaoqi Jiang, Annette Kopp-Schneider, Peter Neuhaus, Marcus Bahra, Bruno Sinn, Roland Eils, Nathalia Giese, Thilo Hackert, Oliver Strobel, Jens Werner, Markus W. Büchler, Wilko Weichert, Andreas Trumpp, Martin R. Sprick. CYP3A5 mediates basal and acquired therapy resistance in different subtypes of pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-120.